peripheral chondrosarcoma progression is accompanied by

The role of EXT and growth signalling pathways in osteochondromaand its progression towards secondary peripheral chondrosarcomaHameetman, L.

CitationHameetman, L. (2007, April 26). The role of EXT and growth signalling pathways inosteochondroma and its progression towards secondary peripheral chondrosarcoma.Department Pathology, Faculty of Medicine / Leiden University Medical Center (LUMC),Leiden University. Retrieved from https://hdl.handle.net/1887/11865 Version: Corrected Publisher’s Version

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Decreased IHH signalling in peripheral chondrosarcomas

Liesbeth Hameetman, Leida B. Rozeman,Marcel Lombaerts, Jan Oosting,

Antonie H.M. Taminiau,Anne-Marie Cleton-Jansen,

Judith V.M.G. Bovée,Pancras C.W. Hogendoorn

Peripheral chondrosarcomaprogression is accompanied bydecreased Indian Hedgehog(IHH) signalling 5

Journal of Pathology(2006); 209(4): 501-511

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AbstractHedgehog (HH) signalling is important for specific developmental processes, and aberrant,increased activity has been described in various tumours. Disturbed HH signalling has alsobeen implicated in the hereditary syndrome, Multiple Osteochondromas. Indian Hedgehog(IHH), together with parathyroid hormone-like hormone (PTHLH), participates in theorganization of growth plates in long bones. PTHLH signalling is absent in osteochondromas,benign tumours arising adjacent to the growth plate, but is reactivated when these tumoursundergo malignant transformation towards secondary peripheral chondrosarcoma. We describea gradual decrease in the expression of Patched (PTCH) and glioma-associated oncogenehomolog 1 (GLI1) (both transcribed upon IHH activity), and GLI2 with increasing malignancy,suggesting that IHH signalling is inactive and PTHLH signalling is IHH independent in secondaryperipheral chondrosarcomas. cDNA expression profiling and immunohistochemical studiessuggest that transforming growth factor-β (TGF-β)-mediated proliferative signalling is activein high-grade chondrosarcomas since TGF-β downstream targets were upregulated in thesetumours. This is accompanied by downregulation of energy metabolism-related genes andupregulation of the proto-oncogene jun B. Thus, the tight regulation of growth plateorganization by IHH signalling is still seen in osteochondroma, but gradually lost duringmalignant transformation to secondary peripheral chondrosarcoma and subsequentprogression. TGF-β signalling is stimulated during secondary peripheral chondrosarcomaprogression and could potentially regulate the retained activity of PTHLH.

Keywords: Hedgehog; transforming growth factor-β; cartilaginous tumours; molecularpathways; tumour progression

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IntroductionHedgehog (HH) signalling plays an important role during embryonic and postembryonicdevelopment, where it regulates cell proliferation and/or differentiation 1. Upon binding toHH, Patched (PTCH) relieves its inhibition of Smoothened (SMO), which activates GLItranscription factor family members (GLI1-3). This leads to activation of target genes, includingGLI1 and PTCH itself 2,3. In the growth plate, Indian Hedgehog (IHH) regulates chondrocyteproliferation and differentiation in a tightly regulated paracrine feedback loop, together withparathyroid hormone-like hormone (PTHLH or PTHrP; figure 5.1A) 4,5,7.

Deregulated IHH signalling has been implicated in patients with Multiple Osteochon-dromas 8-10, an autosomal dominant disorder characterized by the formation of cartilage-capped, benign, bony neoplasms on the outer surface of bones preformed by endochondralossification 9,11. EXT1 and EXT2 have been identified as tumour suppressor genes for MultipleOsteochondromas 12. These genes are involved in the biosynthesis of heparan sulphateproteoglycans (HSPGs) 13,14, multifunctional macromolecules involved in the diffusion of HHto PTCH 10,15. Osteochondromas also occur as solitary lesions in a non-hereditary background11.The cartilage cap of osteochondromas morphologically recapitulates the epiphyseal growthplate 11.

A small fraction (<3%) of osteochondromas transforms into so-called secondaryperipheral chondrosarcoma 16,17, which are classified histologically into three grades thatcorrelate with prognosis 18. Condrosarcomas can recur at a higher histological grade 16,suggesting progression in malignancy with time.

Recent findings have linked upregulated HH signalling to various human diseases 2,such as Gorlin syndrome, in which HH signalling is constitutively active as a result of inactivatingmutations in PTCH 19. Moreover, constitutively active HH signalling has been found in severalsporadic cancers, including sporadic basal cell carcinoma 20, medulloblastoma 21, colon cancer22,small-cell lung cancer 23 and prostate cancer 24. Inactivation of HH signalling has beenassociated with developmental malformations such as cyclopia and holoprosencephaly 2.

The involvement of IHH signalling in tumourigenesis as well as endochondral ossificationpoints to its possible involvement in osteochondroma and chondrosarcoma. We have previously

demonstrated that molecules downstream in the IHH/PTHLH signalling pathway are notexpressed in osteochondromas 25,26, suggesting that growth signalling is disturbed. Malignanttransformation leads to re-expression of these signalling molecules 25,26.

Here, we aimed at elucidating the role of IHH signalling in the progression of peripheralchondrosarcomas, in a series of hereditary and sporadic osteochondromas, as well aschondrosarcomas. The same series was subjected to genome-wide expression analysis toidentify other signal transduction pathways involved in chondrosarcoma progression.

Material and methodsPatient materialFresh frozen samples were obtained from resected specimens (table V.I). Growth plateswere acquired from resections or biopsies for orthopaedic clinical conditions not related toosteochondroma or chondrosarcoma. All tissue samples were handled in a coded fashion,according to Dutch national ethical guidelines (“Code for Proper Secondary Use of HumanTissue”, Dutch Federation of Medical Scientific Societies).

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RNA isolationRNA was isolated from tumour samples that contained at least 70% tumour cells, as determinedby haematoxylin and eosin stained frozen sections, as described previously 27. From L-493,RNA was isolated from two different parts of the tumour (one part corresponding to a low-grade area and the other with high-grade morphology), to investigate possible differences ingene expression within one tumour.

Quantitative reversed transcriptase PCR (qPCR)For first strand cDNA synthesis, 1 µg of total RNA was reverse transcribed using AMV reversetranscriptase (Roche, Penzberg, Germany) with 100 ng primer (dT) 15 (Roche) and 50 ngrandom primers (Invitrogen, Carlsbad CA, USA), according to the manufacturer’s instructions.

qPCR reactions were performed as previously described 28 with PCR primers providedin supplementary table V.I. Expression levels were normalized to four genes (CPSF6, GPR108,CAPNS1, and SRPR) selected from expression profiling experiments of peripheral and central29

cartilaginous tumours, with the least variation between all samples using the geNormprogramme 30. Log2 transformed normalized data were analysed in SPSS 11.0 (SPSS Inc.,Chicago, IL, USA). Expression levels in human growth plates were compared with those inosteochondromas, low-grade (grade I) and high-grade (grade II and III) chondrosarcomasusing one-way ANOVA with Bonferroni correction. Spearman’s non-parametric correlationcoefficients were computed for relations between expression levels and histological grade.Corrected p-values ≤ 0.05 were considered significant.

cDNA microarraysA custom-made cDNA microarray was used in order to include more genes involved inchondrogenesis. The list of cDNA clones is available upon request. Array production,hybridization and image acquisition procedures were performed as described previously 29.In brief, tumour and growth plate samples were hybridized against a common referencepanel of cell lines 29,31. For hybridization, 1 µg of sample total RNA and 1 µg of reference paneltotal RNA were used to generate biotin- and fluorescein-labelled cDNA, using the MicromaxTSA labelling kit (Perkin Elmer, Wellesley, MA, USA). Hybridized slides were scanned with aGeneTac LSV scanner (Genomic Solutions, Ann Arbor, MI, USA). Experimental quality waschecked by labelling and hybridizing five samples in duplicate, either as experimental duplicates(n=2) or dye swaps (n=3).

Data analysisSignal intensities were recorded and quantified with Genepix Pro 4.1 software (AxonInstruments, Union City, CA, USA). A Microsoft Excel macro 29 was created to select bonafide spots systematically, normalize these by dividing by the median of all bona fide spotsand log transform the normalized spots.

Analysis was performed for genes expressed in at least 70% of samples. Unsupervisedhierarchical clustering (with “complete linkage” and “correlation”) was performed with SpotfireDecisionsite software for functional genomics (Somerville, MA, USA). Two methods forgroup comparison were used. The first method (corrected t-test method) used the log-transformed ratios of the Excel macro. P-values were calculated by a two-sided Student’s

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Table V.I. Clinocopathological and tumour data

a GP = growth plate; OC = osteochondroma; PCS = peripheral secondary chondrosarcoma grade I, II or III.b MO = Multiple Osteochondromas.c Array = cDNA microarray; qPCR = quantitative reverse transcriptase PCR experiments.d Samples used for standard curve in the qPCR experiments.

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t-test with unequal variance and corrected for multiple testing using the step-up procedurewith a false discovery rate of 10% 32. The second method was the Limma (Linear models forMicroarray Data) package 33, because of its excellent performance in microarray analysis.Limma used the raw Genepix data for within-array print-tip loess normalization of intensitiesafter the same criteria for spot selection were applied, as described in the Excel macro 29.Identities of differentially expressed, spotted clones were verified by sequencing the PCRproducts.

Figure 5.1. Schematic represen-tations of signalling pathways ingrowth plate, osteochondromas,and chondrosarcomas. (A) IHH/PTHLH signalling in the postnatalgrowth plate. The IHH/PTHLHfeedback loop is confined to thegrowth plate. In the transitionzone, IHH will bind PTCH, relievingits inhibitory effect onSmoothened (SMO). SMO thenactivates the transcription factorGLI2, which subsequently movesto the nucleus to start transcrip-tion of IHH target genes. One ofthese targets is PTHLH. AfterPTHLH binds to its receptorPTHR1, BCL2 is upregulated,inhibiting chondrocyte differen-tiation (adapted from Amling etal.4, Van der Eerden et al. 5, Mo etal. 6 and Hogendoorn et al. 7). IfEXT proteins are defective orabsent, HSPG expression at thecell surface may be altered orabsent, affecting the diffusion ofIHH to its receptor. IHH signallingmolecules are indicated in red,PTHLH signalling molecules inblue, and molecules investigatedby quantitative reversed trans-criptase polymerase chain reac-tion (qPCR) by *. (B) Schematicrepresentation of changes inextracellular matrix, cellularity,and the expression of signallingpathways in the course ofmalignant transformation ofosteochondromas and subsequentprogression of peripheral chondro-sarcoma. The different tumourtypes are represented by their histology. Malignant transformation of osteochondromas and furtherprogression of peripheral chondrosarcoma are characterized by a decrease in chondroid matrix and anincrease in cellularity, and the strict organization seen in growth plate is lost. In osteochondromas, there isIHH, canonical WNT and TGF-β signalling but no PTHLH signalling. During malignant transformation oftumour cells that have escaped from the tight control of IHH, PTHLH signalling is activated, but WNTsignalling decreases. During the progression of grade I towards grade III lesions, IHH signalling graduallydiminishes and WNT signalling is lost. The proliferative TGF-β signalling pathway is upregulated duringprogression and activates target genes needed to acquire a more malignant phenotype.

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Differentially expressed genes were selected for verification by either qPCR orimmunohistochemistry. The micro-array series could be expanded with seven extra tumours(table V.I) for qPCR analysis.

ImmunohistochemistryExpression of JUNB (antibody sc-8051, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA,dilution 1:150) and PAI1 (SERPINE1 protein; antibody #3785, American Diagnostica, Stanford,CT, USA, dilution 1:350) was evaluated in 18 osteochondromas, 39 chondrosarcomas, and12 human growth plates. Staining and scoring of 4-µm sections of formalin-fixed, paraffin-embedded material was performed as described 25. Staining intensity was compared withinternal positive controls: osteoblasts and osteoclasts for JUNB, blood vessels and connectivetissue for PAI1.

Activity of TGF-β signalling and WNT-signalling was investigated by presence of eithernuclear phosphorylated Smad2 (PS2 antibody, kindly donated by P. ten Dijke 34, dilution1:2000) expression or nuclear β-catenin (clone 14, BD Biosciences, Erembodegem, Belgium,dilution 1:800) expression. Osteoblasts served as positive internal control for PS2, andosteoblasts and blood vessels for β-catenin.

A χ2 test and Spearman’s non-parametric correlation were used in the statisticalanalysis. Values of P ≤ 0.05 were considered significant.

ResultsqPCR to detect mRNA expression of IHH signalling moleculesExpression of seven genes involved in IHH signalling (figure 5.1A) was assayed using qPCR,and compared to human growth plates (table V.II). Compared with growth plates, PTCHexpression was decreased in low-grade (p = 0.039) and high-grade chondrosarcomas (p =0.001) but not in osteochondromas (p = 1.00; figure 5.2A). Of the GLI transcription factors,GLI2 had the highest expression in the growth plate (p = 0.006) and was reduced in low-grade chondrosarcomas (p = 0.015). Expression of both GLI2 and GLI1 was even lower inhigh-grade tumours compared to growth plate (p = 0.002 and p = 0.043, respectively;

figure 5.2B-C). GLI1 expression correlated with GLI2 expression (r = 0.584, p = 0.02). GLI3expression was not affected in tumours (table V.II).

Figure 5.2. qPCR results of IHH signalling molecules.(A-C) Log-transformed relative mRNA expressionlevels in growth plate, osteochondromas, and low-grade and high-grade chondrosarcomas represented inboxplots of (A) PTCH expression, (B) GLI2 expression, and (C) GLI1 expression. *Significantly lower expressioncompared to growth plate (p < 0.05), Diamonds=extreme values; filled circles=outliers; PCS=peripheralchondrosarcoma

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Increasing histological grade correlated negatively to the expression of PTCH(r = -0.600, p = 0.001), GLI1 (r = -0.458, p = 0.018), and GLI2 (r = -0.457, p = 0.019),suggesting gradual downregulation of IHH signalling during tumour progression (figure 5.1B).Expression of PTCH (r = -0.810, p < 0.001), GLI1 (r = -0.524, p = 0.006), and GLI2(r = -0.421, p = 0.032) correlated negatively with patient age (supplementary figure 5.1),gradually diminishing over 40 years. Using age as a covariate in the analysis, the differencein expression between growth plates and chondrosarcomas was only significant for GLI2 (p= 0.034).

For the grade II chondrosarcoma L-493, two separate tissue samples were used.Expression data from the matrix-rich sample were similar to those of low-grade tumours,while results from the more cellular and myxoid sample corresponded to those of high-gradechondrosarcomas.

PTHLH mRNA expression was present in all tumours. PTHLH protein expression data,as determined previously 25, was available for four osteochondromas and 14 chondrosarcomas,and correlated with the mRNA expression (r = 0.53, p = 0.01).

Expression profilingTo identify signal transduction pathways that might be alternatives to IHH signalling, genome-wide cDNA microarray analysis was performed on 20 tumours and four growth plates.Hierarchical cluster analysis showed that technical duplicates and dye swaps cluster together(supplementary figure 5.2), assuring technical reproducibility and absence of dye bias aswell as the microarray quality. Duplicate spotted clones showed optimal correlation as well.The samples from L-493 with different histology did not cluster together, in contrast totechnical duplicates from other samples, complying with aforementioned qPCR results.Unsupervised clustering showed no distinct clusters of tumour samples, indicating that thedifferent histological grades are characterized by more subtle changes in gene expression.Despite the good overall quality of the microarray, 20 cDNA clones of genes involved in IHHand PTHLH signalling performed suboptimal and could therefore not be optimally correlatedto the qPCR results of these genes.

Table V.II. Log2 transformed relative expression data of IHH signalling molecules in qPCR

a PCS = peripheral chondrosarcoma.b Median = median log transformed expression level of the group.c P-value after Bonferroni correction.

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Limma analysis revealed 17 differentially expressed cDNA clones betweenosteochondromas and human growth plates (supplementary table V.II). Fourteen cloneswere more highly expressed in osteochondromas, including multiple clones encoding theproto-oncogene jun B (JUNB) and several metallothionein 1 (MT1) genes.

We did not find any significantly differentially expressed genes in the comparison ofosteochondroma and grade I chondrosarcoma.

A comparison of grade I and grade III chondrosarcomas for analysis of tumourprogression identified 79 differentially expressed genes with the corrected t-test methodand 32 with the Limma package (supplementary table V.III). Eleven genes were present inboth analyses. Several genes involved in TGF-β signalling were upregulated in grade IIIchondrosarcomas: fibronectin 1(FN1); serine (or cysteine) proteinase inhibitor, clade E (nexin,plasminogen activator inhibitor type 1), member 1 (SERPINE1); thrombospondin 1 (THBS1);and cyclin-dependent kinase inhibitor 1A (CDKN1A, p21, CIP1). TGF-β1 was upregulated inhigh-grade chondrosarcomas (supplementary table V.III). Several genes involved inextracellular matrix (ECM) remodelling were upregulated; among the downregulated genes(corrected t-test n=23; Limma n=5) were those encoding the α1 chains of type IX collagen(COL9A1) and type II collagen (COL2A1). Additionally, several genes encoding proteinsinvolved in oxidative phosphorylation were downregulated in grade III chondrosarcomas.These included genes encoding NADH dehydrogenase (ubiquinone) 1 β subcomplex, 8(NDUFB8), a component of complex I, and cytochrome c oxidase subunit 8A (COX8A) ofcomplex IV. Also, glycolytic enzyme aldolase C (ALDOC), part of the glycolysis, wasdownregulated in grade III chondrosarcomas compared with grade I lesions.

qPCR verification of cDNA microarray resultsTo confirm the differences in mRNA expression of genes relevant to TGF-β signalling,extracellular matrix and oxidative phosphorylation found in the progression analysis, weperformed qPCR on four genes representing these groups (table V.III). The qPCR results forFN1, PLOD3 and NDUFB8 correlated with the microarray expression data (p = 0.006, p =0.004 and p = 0.052 respectively). No correlation was found for COX8A (p = 0.948). FN1was upregulated in grade III chondrosarcomas compared with osteochondromas, grade I

a PCS = peripheral chondrosarcomab Correlation of qPCR expression data with microarray expression data available for four growth plates, fourosteochondromas, eight grade I, five grade II and two grade III chondrosarcomas

c Median = median log transformed expression level of the group

Table V.III. Log2 transformed normalized expression levels for verification microarray data

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and II chondrosarcomas (p = 0.011, 0.014, and 0.012, respectively). Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3) expression was significantly positively correlatedwith increasing histological grade (r = 0.506, p = 0.008), while NDUFB8 showed a negativecorrelation (r= -0.391, p = 0.049). Additionally, mRNA expression of NADH dehydrogenase(ubiquinone) Fe-S protein 3 (NDUFS3) of oxidative phosphorylation complex I, and aldolaseA (ALDOA) of glycolysis were analyzed. Similar to NDUFB8, NDUFS3 expression was negativelycorrelated with tumour progression (r = -0.529, p = 0.005). Expression of COX8 and ALDOAdecreased with progression. ALDOA was upregulated in grade I chondrosarcomas whencompared with osteochondromas (p = 0.024).

Figure 5.3. Immunohistochemical results. (A) Low-power view of an osteochondroma with weak PAI1protein staining in less than 25% of the cells. Note the positive blood vessels, used as internal positivecontrol; (B) a grade II chondrosarcoma with strong cytoplasmic PAI1 protein staining in 75-100% of thecells; (C) Boxplot of total scores from the semiquantitative scoring of PAI1 protein expression. There is asignificant positive correlation between progression and PAI1 protein expression (r = 0.447, p < 0.001).(D) Low-power view of an osteochondroma demonstrating weak nuclear JUNB protein expression in 25-50% of the cells; (E) grade I chondrosarcoma with moderate nuclear JUNB protein expression in 75-100%of the cells; (F) Strong nuclear JUNB expression was found in most cells in this grade II chondrosarcoma.(G) Low-power view of a human epiphyseal growth plate with strong nuclear and cytoplasmic phosphorylatedSmad2 staining in the proliferating and hypertrophic chondrocytes, whereas the resting chondrocytes arenegative; (H) Peripheral chondrosarcoma grade II with strong phosphorylated Smad2 expression in themajority of tumour cells. The same was observed in grade III chondrosarcomas (I).

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Table V.IV. Immunohistochemistry results of PAI1 and JUNB

a Number of positive tumours/total number of tumours that could be evaluated. Data for thetumours for which cDNA array analysis was also available are shown in parentheses.

b Five osteochondromas and nine chondrosarcomas could not evaluated because the internalpositive controls were negative.

Immunohistochemical analysis of protein expressionSERPINE1 and JUNB microarray expression data were verified by immunohistochemistry(table V.IV).

The sum score of PAI1 (SERPINE1 protein) correlated with the mRNA expression(r= 0.488, p = 0.047) and with increasing histological grade (r = 0.447, p < 0.001, figure5.3A-C).

For 16 samples (two human growth plates, three osteochondromas, and five grade I,four grade II, and two grade III chondrosarcomas), also included in the microarrayexperiments, JUNB protein expression correlated with the mRNA expression (r = 0.544,p = 0.016). Expression analysis revealed differential expression of JUNB mRNA betweengrowth plate and osteochondroma. When comparing growth plates and osteochondromasfor JUNB protein expression, no difference was observed (Fisher’s Exact, p = 0.179). Thenumber of JUNB-positive tumours significantly correlated with increasing histological grade(χ2 , linear-by-linear, p = 0.005, figure 5.3D-F, table V.IV).

In human growth plates, both cytoplasmic and nuclear expression of phosphorylatedSmad2 was observed in proliferating and hypertrophic chondrocytes (figure 5.3G). Expressionwas variable in resting chondrocytes. Nuclear and cytoplasmic expression of phosphorylatedSmad2 was observed in all osteochondromas and chondrosarcomas (figure 5.3H-I), implicatingactive TGF-β signalling (figure 5.1B).

Nuclear expression of β-catenin, was found, 9/17 (53%) osteochondromas, 4/21(19%) grade I chondrosarcomas, and in the hypertrophic zone of 5/12 (42%) human growthplates, but not in 11 grade II and two grade III chondrosarcomas. This indicated that canonicalWNT signalling decreased with increasing malignancy (χ2 p = 0.020; figure 5.1B). Twenty-two tumours also included in the frozen series demonstrated a positive correlation betweenPTCH expression and nuclear expression of β-catenin (r = 0.567, p = 0.006).

DiscussionThis study shows that mRNA expression of IHH downstream targets is gradually downregulatedduring tumour progression in peripheral chondrosarcoma compared with normal growthplates, from which its benign precursor, osteochondroma, originates. Expression of bothPTCH and GLI1, two genes transcribed upon activation of HH signalling 3, negatively correlated

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with increasing histological grade. This was also found for GLI2, which transduces the IHHsignal during endochondral bone development 6, but is not a known downstream target ofIHH. Recently, GLI3 has been identified as a key effector of IHH signalling during cartilagedevelopment 35, where IHH antagonises the repressor activity of GLI3 on PTHLH expressionand proliferation. However, in chondrosarcomas GLI3 expression was similar to the expressionseen in human growth plate (table V.II). Therefore, there was no indication that GLI3 playsa role in chondrosarcoma progression.

Recently, constitutively active IHH signalling was demonstrated in chondrosarcomaexplants 36. These contradictory results could be partly explained by the selection ofhousekeeping genes for qPCR analysis. This has been shown to be tissue type specific 30,which led us to choose genes based upon the expression array results, instead of choosingrandom, non-tumour-specific genes, like GAPDH, which was not stably expressed incartilaginous tumours. Also, no discrimination was made by Tiet et al. between peripheraland central-type chondrosarcomas 36, even though it was previously shown that these subtypesclearly have a different genetic make-up 37,38 and other distinct pathways might be operative.

IHH signalling has been shown to be upstream of canonical WNT signalling cascadesrequired for osteogenic differentiation 39. WNT signalling appears to be involved in chondrogenicdifferentiation, since we could demonstrate decreased nuclear expression of β-catenin duringchondrosarcoma progression (figure 5.1B). This intriguing potential relation between IHHand WNT signalling in chondrogenesis needs further studies.

EXT1 and EXT2 are of importance for the diffusion of IHH to its receptor 10,15,40.Consequently, there is a strong indication that IHH signalling is important in osteochondromaformation. Here we have demonstrated that the expression of IHH signalling in osteo-chondromas did not differ from the expression found in growth plates. Morphologically,osteochondromas strongly resemble the epiphyseal growth plate 11, and different zones ofendochondral ossification can sometimes be distinguished. It was technically not possible todissect these different layers to investigate zone-specific expression of IHH signallingmolecules. This probably also affects expression levels of genes that are only expressed in aspecific layer.

Similar to growth plates, osteochondromas cease to grow after puberty 11. Thereforewe investigated the putative relationship between mRNA levels of IHH signalling moleculesin tumours and patient age, and demonstrated decreasing expression of PTCH, GLI1, andGLI2 over a period of 40 years. However, the observation that diagnosis is correlated withage 41 and the distinct expression profiles of low-grade and high-grade tumours of patientswith the same age (including the two samples from L-493), suggest that expression of PTCH,GLI1, and GLI2 does correlate with tumour progression.

Despite inactivation of IHH signalling, there is active, IHH-independent PTHLH signallingin chondrosarcomas (figure 5.1B) 4,25. This is similar to what was found in centralchondrosarcomas 28, but not to murine growth plate, where IHH has been shown to directlyregulate PTHLH expression 42. Our results showed a discrepancy between PTHLH mRNA andpreviously published protein expression 25. However PTHLH mRNA has a very short half-life43, making it difficult to correlate protein and gene expression.

A good candidate to activate PTHLH signalling in the absence of IHH is TGF-β, whichcan regulate PTHLH expression independent of IHH 44. Genome-wide expression profiling

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experiments revealed that tumour progression was indeed associated with upregulation ofgenes involved in TGF-β signalling, including TGF-β1, comparable to results found in othertumours 45. Expression of FN1, SERPINE1, and THBS1 was upregulated in grade IIIchondrosarcomas (tables V.III and V.IV). These genes are known downstream targets ofTGF-β signalling and significant in regard to tumour invasion and metastasis 46. Upregulationof the TGF-β downstream cell cycle inhibitor CDKN1A has previously been noted at theprotein level 25. The changes observed in extracellular matrix-related genes could also beregulated by TGF-β. Taken together, these results implied that TGF-β signalling is activated inhigh-grade chondrosarcomas and this conclusion was supported by the presence of nuclearphosphorylated Smad2 in these tumours. Phosphorylated Smad2 was also present inosteochondromas and human growth plates. For the latter the expression pattern was similarto that of unphosphorylated Smad2 in rat epiphyseal growth plate 47. One can hypothesizethat in chondrosarcomas TGF-β activates specific target genes needed to acquire a malignantphenotype. The possibility cannot be excluded that some of these differentially expressedgenes, like THBS1 and FN1, are the result of the increased vascularization in high-gradechondrosarcomas 48,49.

The diminished amounts of chondroid matrix in high-grade chondrosarcomas 16 andits lack of cellular organization in comparison with osteochondroma suggest loss of the IHH/PTHLH feedback loop (figure 5.1). IHH signalling is a tightly controlled signalling pathway.We observed that osteochondroma cells are still controlled by IHH and undergo endochondralossification. As the osteochondroma ages, IHH signalling decreases, which was also seen inthe growth plate 50, and all tumour cells will eventually differentiate. One can hypothesizethat tumour cells may escape from IHH control to transform into a malignancy, by switchingto less controlled proliferative signalling pathways, such as TGF-β signalling, thereby causinga cascade of events resulting in tumour progression (figure 5.1B). To further explore thishypothesis, a model system would be helpful, but this is hampered by differences betweenhuman and rodents, in which there is no closure of the growth plate at the end of sexualmaturation 51.

Further analysis of the genome-wide expression studies revealed decreased expression

of genes that encode proteins involved in oxidative phosphorylation and glycolysis in gradeIII chondrosarcomas compared to grade I chondrosarcomas. Glycolysis is upregulated inhypoxic environments, such as paucivascular osteochondromas and low-grade chondro-sarcomas 11,16, giving cells a growth advantage 52. High-grade chondrosarcomas show increasedvascularisation 48,49, which will raise oxygen availability and thereby abolish the need forglycolysis. This was reflected by the downregulation of ALDOA and ALDOC in grade IIIchondrosarcomas.

Oxidative phosphorylation was simultaneously downregulated with glycolysis. Inotherwise non-invasive C2C12 myoblasts, depletion of mitochondrial DNA induces an invasivephenotype 53 and increases resistance to apoptosis, in part by up-regulating BCL2 54. High-grade chondrosarcomas have high BCL2 expression 25 and an increased risk of recurrenceand metastasis 16, consistent with an invasive phenotype.

The expression profiles of osteochondromas and grade I chondrosarcomas wereindistinguishable. This distinction is also considered difficult at the histological level and isusually based on a combination of histological, radiological, and clinicopathological data 16.

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Most likely the differences in gene expression are very subtle or only detectable at theprotein level and cannot be detected with the method used.

Both qPCR and genome-wide expression profiling revealed similarities in geneexpression between osteochondromas and human epiphyseal growth plates. However, JUNB(and to lesser extent FOSB) were more highly expressed in osteochondromas than in growthplates. JUNB and FOSB are members of the AP-1 transcription factor family, which has beenimplicated in endochondral ossification in mice 55,56. Remarkably, we found very low levels ofJUNB mRNA and protein in growth plates. JUNB can have a stimulatory effect on chondrocyteproliferation 55. At the protein level, JUNB gradually increased during malignant transformationand further progression, which is consistent with its effect on proliferation. This increase inJUNB expression might also be regulated by TGF-β 57.

In conclusion, IHH signalling controls the tight regulation of growth plate organizationand is still active in osteochondroma. However, IHH signalling is gradually inactivated duringperipheral chondrosarcoma progression when tumour cells adapt to a more malignantphenotype (figure 5.1B). TGF-β signalling can potentially regulate PTHLH signalling andconcurrent remodelling of the extracellular matrix. Downregulation of genes involved inoxidative phosphorylation and glycolysis accompanies the more invasive phenotype of high-grade tumours.

AcknowledgementsWe thank P ten Dijke for providing the PS2 antibody, A Yavas, PM Wijers-Koster, HJ Baelde,and HJ van Paassen for technical assistance; P Eilers for his help with statistical analysis; Rvan Eijk and T van Wezel for their assistance with the cDNA array experiments; and S Romeofor fruitful discussions. This study was financially supported by the Dutch Cancer Society(grand number: RUL 2002-2738), and presented at the 18th Annual Meeting of the EuropeanMusculoSkeletal Oncology Society (EMSOS), where it was awarded the EMSOS prize. Thedepartment of Pathology, LUMC is partner of the EuroBoNeT consortium, a EuropeanCommission granted Network of Excellence for studying the pathology and genetics of bonetumours.

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Supplementary material

a Conditions are available upon request

Supplementary figure 5.1. Correlation between expression of IHH signalling and patient’s age. Logtransformed relative mRNA expression levels in growth plate, osteochondromas, low-grade and high-gradechondrosarcomas of (A) PTCH, (B) GLI2 and (C) GLI1 expression. The expression is related to the patient’sage. The Pearson correlation is given with corresponding p-value. All three genes have a negative correlationwith age. The two samples of L-0493 are circled.

Supplementary table V.I. Genes investigated by quantitative PCR analysis

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Supplementary figure 5.2. Dendogram of unsupervised cluster analysis.Unsupervised clustering of 3074 gene expression ratios for cDNA that gaveinterpretable results in at least 70% of the samples. The duplicate (du) and dyeswap (ds) cluster together with their original sample.GP: growth plate; OC: osteochondromas; mo: Multiple Osteochondromas; PCS:peripheral chondrosarcoma.

Supplementary table V.II. Results Limma analysis osteochondroma vs growth plate

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Supplementary table V.III. Genes differentially expressed between grade I and grade III chondrosarcoma

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Supplementary table V.III continued

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Supplementary table V.III continued

a The genes found in both analyses are indicated in boldb FDR: False discovery rate < 0.1 (10%)c B: log-odds that a gene is differentially expressed

peripheral chondrosarcoma progression is accompanied by

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